Heparan sulfate (HS) is a highly sulfated polysaccharide with important roles in many physiological and pathological events. The structural diversity of naturally existing HS, which exhibits diverse sulfation patterns, has hindered the understanding of its structure-activity relationships. To date, synthetic methodologies toward HS are mostly target oriented, leading to a small set of oligosaccharides. To enable a deeper understanding of HS biology, the availability of large collections of HS oligosaccharides is critical. Glycan Therapeutics is a company specialized in HS-related research products. In this project, Glycan Therapeutics will collaborate with leading experts in HS synthesis to produce and commercialize large libraries of HS oligosaccharides. In Aim 1, the synthesis of key disaccharide building blocks will be expedited. Rather than starting from monosaccharides, new methods have been developed to hydrolyze natural heparin and heparosan polysaccharides to obtain disaccharides. These disaccharides can then be transformed into suitably protected building blocks ready for glycosylation and glycan chain extension. Notably, this process reduces the total number of synthetic steps needed to produce the disaccharide building blocks by ~50%. In Aim 2, a comprehensive library of HS tetrasaccharides bearing all possible 2-O, 6-O and N-sulfation sequences will be synthesized. A new protective group strategy has been designed that will enable 256 HS tetrasaccharides to be accessed from only four strategically protected tetrasaccharides by automated solid- phase synthesis. All tetrasaccharides are functionalized to facilitate bioconjugation and microarray studies. In Aim 3, a library of 13C-labeled oligosaccharide standards carrying a ?UA4,5-unsaturated residue at the non- reducing end will be synthesized. A common method for researchers to determine HS composition is to use heparin lyases to degrade HS into disaccharides, followed by disaccharide composition analysis using LC-MS. Currently, there are no commercial sources for stable isotopically-labeled 3-O-sulfated standards. Glycan Therapeutics will synthesize a series of these standards to enable quantitative analysis of HS composition. In Phase I studies, four key strategically protected disaccharides (200 mg each), four protected strategically protected tetrasaccharide precursors (200 mg each), ten fully deprotected, sulfated HS tetrasaccharides (2 mg each), as well as two 13C-labeled and 3-O-sulfated HS oligosaccharides (1 mg each) will be prepared. In Phase II studies, the synthesis will be scaled up to produce the strategically protected disaccharides (10 g each), the 256-member library of HS tetrasaccharides bearing all possible 2-O, 6-O and N-sulfation sequences (2 mg each), and a 13C-labeled and 3-O HS oligosaccharide library (28 compounds, 3 mg each). The new HS structures that will become available from this project will represent the largest, most comprehensive HS libraries to date. Successful commercialization of these products will greatly accelerate research on the chemistry and biology of HS, and facilitate the development of HS-based therapeutics.